Effects of Dietary Cereal and Protein Source on Fiber Digestibility, Composition, and Metabolic Activity of the Intestinal Microbiota in Weaner Piglets

Simple Summary Rye and rapeseed meal can be alternative feed components for weaner piglets instead of wheat and soybean meal. Both components can help to meet current challenges in pig nutrition, such as increasingly dry weather conditions and the high amount of imported soybean. Since they contain more and differently composed fiber, effects on digestive physiology and intestinal microbiota might help to maintain gut health and prevent post-weaning diarrhea. This study shows that despite a similar composition of the large intestinal microbiota, the higher amount and solubility of complex carbohydrates from rye lead to a higher fermentative activity compared to wheat, which is considered a beneficial effect. The high amount of insoluble dietary fiber in rapeseed-based diets lowered bacterial metabolic activity and caused a shift toward insoluble fiber degrading bacteria. Abstract This study aimed to investigate the effect of fiber-rich rye and rapeseed meal (RSM) compared to wheat and soybean meal (SBM) on fiber digestibility and the composition and metabolic activity of intestinal microbiota. At weaning, 88 piglets were allocated to four feeding groups: wheat/SBM, wheat/RSM, rye/SBM, and rye/RSM. Dietary inclusion level was 48% for rye and wheat, 25% for SBM, and 30% for RSM. Piglets were euthanized after 33 days for collection of digesta and feces. Samples were analyzed for dry matter and non-starch-polysaccharide (NSP) digestibility, bacterial metabolites, and relative abundance of microbiota. Rye-based diets had higher concentrations of soluble NSP than wheat-based diets. RSM-diets were higher in insoluble NSP compared to SBM. Rye-fed piglets showed a higher colonic and fecal digestibility of NSP (p < 0.001, p = 0.001, respectively). RSM-fed piglets showed a lower colonic and fecal digestibility of NSP than SBM-fed piglets (p < 0.001). Rye increased jejunal and colonic concentration of short-chain fatty acids (SCFA) compared to wheat (p < 0.001, p = 0.016, respectively). RSM-fed pigs showed a lower jejunal concentration of SCFA (p = 0.001) than SBM-fed pigs. Relative abundance of Firmicutes was higher (p = 0.039) and of Proteobacteria lower (p = 0.002) in rye-fed pigs compared to wheat. RSM reduced Firmicutes and increased Actinobacteria (jejunum, colon, feces: p < 0.050), jejunal Proteobacteria (p = 0.019) and colonic Bacteroidetes (p = 0.014). Despite a similar composition of the colonic microbiota, the higher amount and solubility of NSP from rye resulted in an increased fermentative activity compared to wheat. The high amount of insoluble dietary fiber in RSM-based diets reduced bacterial metabolic activity and caused a shift toward insoluble fiber degrading bacteria. Further research should focus on host–microbiota interaction to improve feeding concepts with a targeted use of dietary fiber.


Introduction
An important approach to stabilize gut health of weaner pigs is the optimization of intestinal microbial colonization in the sense of intestinal eubiosis and beneficial bacterial mucus marker N-acetylneuraminic acid, as well as on composition and metabolic activity of intestinal microbiota.

Animals and Diets
The animal study was described in detail previously [14]. In brief, 88 piglets (German Landrace, bred at the Institute of Animal Nutrition, Freie Universität Berlin, 8.3 ± 1.1 kg body weight) were weaned at day 28 of life and randomly allocated to four feeding groups in a 2 × 2 factorial design with wheat/SBM (W-SBM), wheat/RSM (W-RSM), rye/SBM (R-SBM), and rye/RSM (R-RSM). Piglets were housed with two animals per pen and 11 pens per treatment. The pen was used as statistical unit (n = 11). The health status was monitored daily by controlling general condition, feed intake, and fecal consistency of the piglets. Water and feed were supplied ad libitum during the experimental period of 33 days. The diets were formulated to meet or exceed the recommendations for piglet nutrition [26] and calculated to be isonitrogenous. Wheat (Triticum aestivum) or hybrid rye (Secale cereale) were included as cereals at 48 and 25% of SBM or 30% of RSM as protein rich ingredient. The average concentration of crude protein in the complete diet was 220 g/kg. Crude fiber varied among the groups with 22 g/kg in W-SBM, 52 g/kg in W-RSM, 18 g/kg in R-SBM, and 48 g/kg in R-RSM. The exact feed formulation, results of the nutrient analysis, and digestibility determinations are available elsewhere [14].

Sampling
One piglet per pen was dissected after 33 days of trial duration. Pigs were chosen to achieve balanced numbers of males and females in each treatment group. After anaesthesia by 20 mg of ketamine hydrochloride (Ursotamin, 10%; Serumwerk Bernburg AG, Bernburg, Germany) and 2 mg of xylazine (Xylazin, 2%, Serumwerk Bernburg AG, Bernburg, Germany) per kg body weight, pigs were euthanized with an intracardial injection of tetracaine hydrochloride, mebezonium iodide, and embutramide (T61; Intervet, Unterschleißheim, Germany). The gastrointestinal tract was removed and digesta samples were collected from jejunum, ileum, colon ascendens, and rectum. From each intestinal segment, one subset of digesta samples was stored at −20 • C for analysis of dry matter (DM), fiber and N-acetylneuraminic acid. Another subset was snap frozen with liquid nitrogen and stored at −80 • C for analysis of bacterial metabolites and 16S rRNA sequencing.

Determination of Digesta Content and Apparent Digestibility of DM and NSP
Digesta samples from jejunum, colon ascendens, and feces were dried in an oven at 103 • C overnight to determine the DM content. Colonic and fecal samples were lyophilized and total and insoluble NSP content was measured as described for NSP in the diets [14]. To determine apparent digestibility of DM and NSP, titanium dioxide content was determined as described before [27] in lyophilized and grinded (0.5 mm particle size) digesta, feces, and in the diets. Apparent digestibility of DM and NSP was calculated as follows [28]: Digestibility (%) = 100 − (TiO2 in feed (%))/(TiO2 in digesta/feces (%)) × (Nutrient in digesta/feces (%))/(Nutrient in feed (%)) × 100 Apparent digestibility determined in feces was further considered as apparent total tract digestibility (ATTD).

Analysis of N-Acetylneuraminic Acid as Marker of Intestinal Mucus Production
Lyophilized and grinded samples of ileal digesta were hydrolyzed with acetic acid (2 mol/L). After centrifugation, the supernatant was analyzed for N-acetylneuraminic acid via HPIC with an amperometrically pulsed detector cell (Thermo Fisher Scientific, Waltham, MA, USA) using a commercial standard of N-acetylneuraminic acid as reference substance (Sigma-Aldrich, Taufkirchen, Germany).

Analysis of Bacterial Metabolites
In jejunal and colonic digesta samples and feces, concentration of SCFA, D-and Llactate, and ammonia was analyzed as described before [29]. Briefly, SCFA were determined via gas chromatography (Agilent Technologies 6890N, autosampler G2614A, and injection tower G2613A; Network GC Systems, Böblingen, Germany) using caproic acid as an internal standard. D-and L-lactate were measured by HPLC (Agilent 1100; Agilent Technologies, Böblingen, Germany). Ammonia was analyzed colorimetrically using the Berthelot reaction in microtitration plates at 620 nm in a Tecan Sunrise microplate reader (Tecan Austria GmbH, Grödig, Austria).

DNA Extraction and 16S rRNA Sequencing
To extract total DNA from 0.25 g of jejunal and colonic digesta and feces, a commercial extraction kit (QIAamp PowerFaecal Pro DNA Kit, Qiagen, Hilden, Germany) was used in accordance with the manufacturer's instructions with an additional lysis step at 65 • C. Homogenization was carried out using FastPrep-24 TM 5G (M.P. Biomedicals LLC, Santa Ana, CA, USA) at 6 m/s for 10 min (4 times 5 × 30 s and 15 s pause time). DNA extracts were stored at −30 • C until further analysis. Extracts were subjected to amplicon sequencing using an Illumina NextSeq500 sequencer (LGC, Berlin, Germany) with two 150-base pair reads. After demultiplexing, BBMerge tool [30] was used for combining paired reads. Resulting 16S rDNA sequences were analyzed using QIIME2 pipeline [31] and the SILVA SSU database [32]. Quality control and determination of sequence counts were performed using the DADA2 [33]. Further details were described previously [34]. Indices of bacterial diversity (Richness, Shannon index, and Evenness) were calculated from ASV level data. Principal component analysis of 16S rRNA data was carried out using the online software ClustVis [35].

Statistical Analysis
Statistical analyses were carried out in SPSS 26.0 (IBM, Chicago, IL, USA). The distribution of data was tested by Kolmogorov-Smirnov test. Normally distributed data were analyzed by 2-factorial ANOVA with cereal (CER) and protein meal (PM) as fixed factors. Group differences were assessed by post hoc Tukey test. p-values below 0.05 were considered significant. Pearson's correlation was analyzed between colonic Bacteroidetes and average daily gain (ADG) and between jejunal Proteobacteria and apparent ileal digestibility (AID) of crude protein and total amino acids.

Results
Results of growth performance and fecal score were described in detail previously [14]. In brief, average daily gain, average daily feed intake, feed conversion ratio and final body weight were not influenced by the feeding of rye compared to wheat. RSM in comparison to SBM reduced average daily gain and average daily feed intake in the overall trial period. Fecal score was within the physiological range throughout the trial.

Apparent Digestibility of DM and NSP
Fecal DM digestibility was lower in rye-fed piglets than pigs receiving wheat-based diets (p = 0.035, Table 2). DM digestibility was lower in RSM-pigs than in SBM-pigs in the jejunum, colon and feces (each p < 0.001).
Colonic NSP digestibility (70.7-88.2%) was higher than fecal NSP digestibility (59.5-74.8%) across the four feeding groups. Both sites showed a higher digestibility of soluble than of insoluble NSP and AX. Rye-fed piglets showed a higher colonic and fecal digestibility of NSP, sNSP, glucose and soluble AX (colon: each p < 0.001; feces: p = 0.001, p < 0.001, p < 0.001, p = 0.005, respectively). Compared to SBM, RSM-fed piglets showed a lower colonic and fecal digestibility of NSP and iNSP (each p < 0.001). Fecal digestibility of iNSP was highest in R-SBM-fed pigs and lowest in R-RSM-fed pigs (p < 0.001). Fecal digestibility of insoluble AX was highest in W-RSM and lowest in R-RSM-fed piglets (p = 0.009).

Concentration of N-Acetylneuraminic Acid in the Ileum Digesta
Ileal digesta concentration of N-acetylneuraminic acid and ratio of N-acetylneuraminic acid to titanium dioxide were measured to estimate mucus production in the small intestine. Nor concentrations neither the ratio were affected by the dietary treatments (p > 0.05, Table 3).

Microbial Diversity Indices
Microbial richness was reduced in jejunal digesta of piglets fed rye compared to wheat (p = 0.047; Table 5). Shannon index and evenness were not affected (p > 0.05).

Relative Abundance of Bacterial Phyla, Order, and Genera
Most abundant phyla in jejunum, colon, and feces were Firmicutes and Bacteroidetes (Table 6). In rye-fed pigs, jejunal Firmicutes were higher and Proteobacteria lower than in wheat-fed pigs (p = 0.039, p = 0.002, respectively). Compared to SBM, RSM reduced Firmicutes, and increased Actinobacteria at the three sampling sites (p < 0.050). RSM increased Proteobacteria in the jejunum and Bacteroidetes in the colon (p = 0.019, p = 0.014, respectively). Correlation (r) between the relative abundance of jejunal Proteobacteria and AID of crude protein was 0.119 (p = 0.484) and with AID of total amino acids it was 0.097 (p = 0.605). Correlation (r) between colonic Bacteroidetes and ADG of days 0-33 was −0.106 (p = 0.498) and of days 28-33 −0.302 (p = 0.049). In the jejunum, W-RSM-fed pigs showed a lower relative abundance of Firmicutes compared to the other three groups (p = 0.002).
Principal component analysis showed higher variabilities of the relative abundance of bacterial genera in jejunal digesta (≤23.3%) than in colonic digesta and feces (≤14.2%; Figure 1). Clusters of the experimental diets were overlapping to a high extent.

Fiber Composition of the Experimental Diets
The use of fiber-rich feed components might help to stabilize the intestinal milieu during the post-weaning period. As rye contains more SDF and sNSP [18,36] the rye-based diets used in the current study were characterized by higher concentrations of soluble fiber fractions than the wheat-based diets. Similarly, the high concentrations of iNSP in RSM [18] resulted in a higher concentration of iNSP in the RSM-based diets compared to the SBM-based ones.
AX are the predominant fraction of cereal NSP and consist of a xylan backbone substituted by arabinose to varying degrees [18]. The degree of substitution can be described by the A/X-ratio, which is similar in rye and wheat [18,20], but higher in RSM compared to SBM [18]. Readily fermentable and more soluble AX are usually more substituted and characterized by a higher A/X-ratio [37]. However, solubility and fermentability of AX are reduced with increasing cross-linkages to lignin, ferulic acid or other polysaccharides and by a higher molecular weight of AX [18,38]. As discussed below, intestinal microbiota was shaped differently by the RSM-based diets in the present study compared to SBM. This might be related to the higher dietary and intestinal A/X-ratio of RSM. However, the high

Fiber Composition of the Experimental Diets
The use of fiber-rich feed components might help to stabilize the intestinal milieu during the post-weaning period. As rye contains more SDF and sNSP [18,36] the rye-based diets used in the current study were characterized by higher concentrations of soluble fiber fractions than the wheat-based diets. Similarly, the high concentrations of iNSP in RSM [18] resulted in a higher concentration of iNSP in the RSM-based diets compared to the SBM-based ones.
AX are the predominant fraction of cereal NSP and consist of a xylan backbone substituted by arabinose to varying degrees [18]. The degree of substitution can be described by the A/X-ratio, which is similar in rye and wheat [18,20], but higher in RSM compared to SBM [18]. Readily fermentable and more soluble AX are usually more substituted and characterized by a higher A/X-ratio [37]. However, solubility and fermentability of AX are reduced with increasing cross-linkages to lignin, ferulic acid or other polysaccharides and by a higher molecular weight of AX [18,38]. As discussed below, intestinal microbiota was shaped differently by the RSM-based diets in the present study compared to SBM. This might be related to the higher dietary and intestinal A/X-ratio of RSM. However, the high content of insoluble dietary fiber (IDF) and acid detergent lignin (ADL) in the RSM-based diets indicating a high degree of lignification might have reduced fermentability, as indicated by the reduced concentration of SCFA in RSM-fed pigs. Therefore, A/X-ratio could not be used as an indicator of degradability of AX in the current study.

Apparent Digestibility of NSP
The apparent colonic and total tract digestibility of total and soluble NSP in rye-fed pigs was higher compared to pigs receiving wheat-based diets. A higher ATTD of TDF and arabinose in rye-fed pigs was associated with an increased microbial fermentation in the large intestine [16]. In rye compared to wheat a higher fermentative activity might be related to the higher solubility of rye AX, the higher content of β-glucans and the higher total amount of dietary fiber [16,37]. Therefore, the digestibility of NSP in the current study might have been caused by a higher uptake of fermentable fiber with the rye-based diets, including more sNSP and soluble AX. Moreover, wheat AX might be less degradable, because of cross-linkages to other polysaccharides such as cellulose [37]. Cellulose is a major plant cell wall component, which is highly insoluble, poorly fermentable, and has a higher concentration in wheat than in rye [18]. The increased concentrations of digesta SCFA and the higher relative abundance of major plant cell wall-degrading bacterial species in the jejunum indicate an enhanced bacterial fiber degradation and can further explain the higher digestibility of NSP in rye-fed pigs of the present study.
With respect to NSP digestibility in RSM-fed pigs, values for colonic and total tract digestibility of total and iNSP was lower than in SBM-fed pigs. Similarly, ATTD of neutral detergent fiber (NDF), acid detergent fiber (ADF), carbohydrates, and dietary fiber was lower in other studies investigating the feeding of RSM instead of SBM to pigs [39,40]. However, one of these studies did not show a different ATTD of NSP and cellulose between RSM and SBM [39]. Microbial fiber fermentation and metabolic activity might be reduced as RSM is more lignified and insoluble than SBM [18]. This is reflected in the greater amount of IDF and iNSP in RSM-based diets of the current study and the lower SCFA concentration in jejunum, colon, and feces of RSM-fed pigs compared to SBM. Surprisingly, the present values for colonic digestibility of NSP were higher than ATTD of NSP. In contrast, other studies show an increasing digestibility of NSP and AX in caudal direction [16,38]. However, the values of NSP digestibility were numerically in the same range, and furthermore, the determination of digestibility in feces is susceptible to interference. Bacteria may act as "non-dietary interfering substance" in NSP analytical procedures resulting in overestimated values of NSP concentration that are higher than the true diet derived NSP concentration [41]. Considering the higher number of bacteria in feces compared to colonic digesta and that in pigs and humans bacterial mass represents 40-50% of fecal DM [42,43], the interfering effect would be more pronounced with respect to ATTD of NSP compared to colonic digestibility. Additionally, some bacteria produce exopolysaccharides such as colonic acid (Enterobacteria) or dextran (Lactobacillales), which might have contributed to increased fecal concentration of NSP [44].

Bacterial Metabolites and Composition of the Microbiome
In the current study, compared to wheat, rye-fed pigs had a higher concentration of SCFA in the jejunum and colon. As shown in a study comparing the feeding of an AX-rich diet (65% rye-flakes) with a diet based on wheat flour, complex cereal polysaccharides such as AX are not degraded enzymatically in the small intestine and might therefore promote the growth of SCFA-producing bacteria in the distal parts of the intestine [38]. Another study focused on the feeding of wheat and rye breads to pigs, demonstrating that increased SCFA-production in the small intestine might be related to the more soluble fraction of dietary fiber, which is readily fermentable, whilst insoluble fractions would be degraded more distally [37]. Moreover, rye might cause a higher bacterial production of butyrate due to the structure of AX and the lower content of cellulose [45]. Therefore, the high concentration of TDF and SDF in the rye-based diets of this study may have provided more substrate for growth of SCFA-producing bacteria. Positive effects of SCFA derived from fermentation of dietary fiber on gut and animal health have been widely reviewed, including the use of acetate as an energy source and butyrate as the main fuel for colonocytes and its efficiency against possible pathogens [7,46]. In the present study, only data on digesta concentration of SCFA are available, but SCFA are absorbed rapidly via the gut wall [47]. Rye-derived SCFA might be of systemic use as peripheral blood concentration of SCFA was increased by an AX-rich diet based on rye-flakes compared to a wheat flour-based diet [48]. Despite the increased colonic concentration of SCFA and especially butyrate in the current study, no changes of the microbial community were determined in colonic digesta. This might be related to cross-feeding of AX-degrading bacteria, e.g., between Bifidobacteria and butyrate-producing bacteria, and to an increased abundance of the phosphotransferase system (kl02060) regulating carbohydrate uptake into bacterial cells [22]. Nevertheless, it is likely that rye compared to wheat enhanced the production of bacterial metabolites by providing a higher amount of easier fermentable substrate without a shift in the microbial community.
Compared to SBM-fed pigs, concentration of SCFA was lower in RSM-fed pigs in the jejunum and tendentially in the colon which indicates a lower metabolic activity of the resident microbiota. In contrast, previous studies showed an equal level of SCFA between RSM and SBM [24,25,28]. However, compared to these studies, the inclusion level of RSM and consequently the content of lignin and IDF was higher in the present study. A high degree of lignification may hamper enzymatic as well as microbial fiber degradation in RSM-fed pigs [39]. In combination with the lower digestibility of NSP and DM, this could explain the lower SCFA compared to SBM-fed pigs. Additionally, SCFA might have been absorbed rapidly or used by other microbiota in the sense of cross-feeding [49].
In accordance with a recent meta-analysis that identified Firmicutes, Proteobacteria, and Bacteroidetes as the most abundant phyla of the core microbiome in pigs [50], Firmicutes and Bacteroidetes were the predominant phyla of the pigs in the current study.
In rye-fed piglets of this study, composition of the microbial community was only different to wheat-fed pigs in the jejunum. This was unexpected, because according to other studies, the major impact of rye on the microbial community is expected in the proximal parts of the large intestine [16,23,38]. A higher relative abundance of Firmicutes in the small intestine might be explained by the higher content of sNSP in the rye-based diets, since more insoluble substrate would be degraded further distally [7]. The phylum Firmicutes contains many plant cell wall-degrading species including Clostridium sensu stricto 1 and Terrisporobacter. These two genera are known to ferment complex indigestible plant polysaccharides such as hemicellulose and cellulose [51]. Therefore, the fiber provided by the rye-based diets might have served as a substrate and stimulated Firmicutes' proliferation. Firmicutes contains many butyrate-producers [52], therefore an increased abundance could be considered as a positive effect of the feeding of rye. However, SCFA are absorbed primarily in the caecum and proximal colon, and might not utilized by the host in the same extent in the jejunum [47].
Proteobacteria had a lower jejunal relative abundance in rye-fed pigs of the current study. Many putative pathogens such as E. coli and Salmonella belong to Proteobacteria and a lower presence of Proteobacteria was associated with an increased intestinal barrier function and a higher anti-inflammatory capacity of the local immune system [53].
In contrast to an increased concentration of SCFA in jejunal digesta, the analysis of microbial diversity in rye-fed pigs showed a lower richness compared to wheat. It is possible that the increased production of metabolites might be driven by only a few selected genera of Firmicutes which were more abundant in the rye-fed pigs.
With respect to effects of RSM on the relative abundance of microbiota compared to SBM, the present study resulted in a reduced abundance of Firmicutes at the three sampling sites. As mentioned above, Firmicutes contains many fiber-degrading species and is known to produce SCFA. The lower NSP and DM digestibility together in RSM-fed pigs compared to SBM indicate that the fiber provided by the RSM-based diets was not used as a suitable substrate for fermentation by microbiota, especially fiber-degrading Firmicutes. Consequently, the metabolic activity and growth of microbiota was lower as illustrated by the lower concentration of SCFA and the lower relative abundance of Firmicutes. In line with this, compared to alfalfa meal, wheat bran and pure cellulose containing a higher amount of IDF also reduced the relative abundance of Firmicutes in large intestinal mucosa of suckling pigs [54]. However, another study investigating the feeding of RSM instead of SBM showed an increase of Firmicutes, although the lower inclusion level and lower content of IDF might have prevented an inverse result [55]. A higher ratio of Firmicutes:Bacteroidetes was associated with a reduction of the incidence of diarrhea and infections [55]. Nonetheless, previously published results of the present study showed that the fecal score of the pigs was in a physiological range throughout the trial [14]. Within Firmicutes, the predominant genera Clostridium sensu stricto 1 and Terrisporobacter were also decreased by RSM in the current study. This might explain the reduced concentration of SCFA in RSM-fed pigs because another study showed a strong correlation between the relative abundance of these genera with the production of large amounts of metabolites from plant fiber [51].
In line with another study investigating the feeding of RSM [25], RSM increased the relative abundance of Actinobacteria along the gastro-intestinal tract in the current study. In contrast, another study did not show differing values of abundance of Actinobacteria in RSM-or SBM-fed pigs [55]. Actinobacteria efficiently use hemicellulose and cellulose [52], both mainly insoluble fiber fractions. Despite RSM and SBM containing similar amounts of cellulose [18] the higher inclusion of RSM compared to SBM and the higher content of IDF in RSM-diets might have promoted growth of Actinobacteria in RSM-fed pigs. In the jejunum, Bifidobacterium, a genus belonging to Actinobacteria, was also increased by the feeding of RSM. Bifidobacterium is considered to improve gut health [7,56] and was increased by the feeding of cellulose in another study [57].
As mentioned above, an increased relative abundance of Proteobacteria may be a risk for animal health. Nevertheless, the pigs of both RSM and SBM groups were in a good condition throughout the trial. Proteobacteria contains many proteolytic genera [58]. RSM may lead to a lower AID of protein and amino acids than SBM [59] which also was demonstrated in this study [14]. However, there was no correlation detected between AID of crude protein and total amino acids and the jejunal relative abundance of Proteobacteria. It is likely that the protein was not available as a substrate for growth of Proteobacteria, because IDF in RSM caused an encapsulation in the rigid cell wall and an increased digesta passage rate [39].
In RSM-fed pigs, the increased relative abundance of Bacteroidetes is most likely related to the higher content of IDF than in SBM-based diets. Bacteroidetes as the second most abundant phylum of the gut microbiota in pigs [50] was also increased by resistant starch in humans [60] and by corn bran in pigs [61], both sources of IDF. In contrast, a study investigating the feeding of RSM instead of SBM to pigs showed a lower abundance of Bacteroidetes which was related to the high pectin content [25]. An increased abundance of Bacteroidetes is associated with weight loss in humans, mice [62], and pigs [63]. The negative correlation between abundance of Bacteroidetes and ADG in the last six days of the trial might suggest that the shift towards more colonic Bacteroidetes was connected to the reduced weight gain in RSM-fed pigs.

Conclusions
In conclusion, compared to wheat, the higher amount and solubility of NSP from rye resulted in an increased degradation and fermentation of NSP and in a higher metabolic activity of intestinal microbiota. However, relative abundance of large intestinal microbiota was not different between pigs fed rye and wheat. RSM caused a lower bacterial metabolic activity than SBM. The higher fiber content of RSM-based diets was expected to increase fermentation, but 30% inclusion of RSM might have provided an excess of IDF. Still, RSM lead to a lower abundance of common fiber degraders of the predominant phylum Firmicutes and an increased abundance of IDF degrading Actinobacteria and Bacteroidetes. Further research is needed to better understand host-microbiota interaction and to improve feeding concepts with a targeted use of dietary fiber.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ani12010109/s1, Table S1: Effects of the experimental diets on the relative abundance (%) of bacterial orders, Table S2: Effects of the experimental diets on the relative abundance (%) of bacterial genera.